The present invention relates to production of biological substances such as tissues, cells, vaccines, viruses and the like.
Hitherto, various culturing apparatuses have been proposed with the development of gas culture technique for an incubator, that is, culture technique in a special gas atmosphere. For instance, as described in Japanese Patent Laid-open Publication No. 41,489/76, there is disclosed an implantation propagation method in which a gas-permeable and liquid-impermeable Teflon tube is arranged in an incubator maintained with a special gas atmosphere, seed cells to be cultured are adhered onto the inner surface of the Teflon tube and a culture solution is flowed through the tube. In U.S. patent application Ser. No. 275,642 filed on Jul. 27, 1972, now U.S. Pat. No. 3,839,155, there is described another implantation propagation method employing a propagation apparatus provided with a number of discs arranged perpendicularly and separated from each other in the horizontal direction. A small amount of a culture solution is filled in said propagation apparatus so as to immerse a part of said discs, cells to be propagated are adhered to said plurality of discs, respectively, and the cells are plated and grown by rotating said plurality of discs at a given speed.
In both of the above culturing apparatuses, however, tissues or cells are propagated on the whole surface of an implantation surface by a one-generation culture, and the whole surface area of the implantation surface is large, so that a comparatively large amount of cells are required initially, but such a large amount of cells should be prepared by previous culture and such preparation is considerably troublesome. Furthermore, an apparatus for carrying out the above method is complicated in construction, large in size, and troublesome to control. In addition, it is difficult to sterilize the portion where the cells are implanted.
As another conventional culture method, a roller bottle culture method as shown in FIG. 1 has been proposed. A schematic diagram shown in FIG. 1 will be explained with reference to a block diagram of the culture operation shown in FIG. 2. In the first place, seed cells and a given amount of a culture solution are poured in several culture bottles 2a, 2b, . . . within a clean bench 1 and the bottles are sealed by suitable plugs. Then, these culture bottles 2a, 2b, . . . are transferred to an incubator 3 maintained with a special gas atmosphere and the plugs are removed. Each bottle is placed on a pair of rotating rollers 4a, 4b, . . . and is rotated at a given speed to rotate the cells housed in the culture bottles 2a, 2b, . . . are rotated and stirred with the culture solution and implanted and propagated on the inner walls of the culture bottles 2a, 2b, . . . After completing predetermined propagation in the incubator 3, the culture bottles 2 a, 2b, . . . are again transferred into the clean bench 1 after sealing the bottles with plugs, where the propagated cells are reimplanted in new culture bottles.
In order to reimplant the propagated cells in new culture bottles, it is firstly necessary to throw away the culture solution in the culture bottles 2a, 2b, . . . which completed the predetermined propagation, and the cells implanted and propagated on the inner wall of the culture bottle are washed by injecting a buffer solution. Then, the buffer solution is thrown away, an enzyme solution such as trypsin or the like is injected and the implanted and propagated cells are made to be easily peeled off the bottle wall. After that, the enzyme solution is thrown away and a new culture solution is injected. Then, the solution is repeatedly sucked and discharged by means of a pipette so that the propagated cells are peeled off the bottle wall and separated into individual cells which are stirred and suspended in the culture solution, and the cell-suspending solution is further divided into one or more new culture bottles.
In the culture method shown in FIG. 1, the desired amount of cells are obtained by repeating reimplantation and roll culture as described above, but in order to reimplant cells, it is necessary to replace the cell-suspending solution into a culture bottle of larger capacity than the culture bottle which just completed propagation or a plurality of culture bottles of equal capacity to the former culture bottle are prepared and the cell-suspending solution is separately injected into these bottles in a fixed quantity. In such method, therefore, the culture operation is troublesome, and there is the possibility of contaminating the cells at the time of reimplantation. Further, it is necessary to circulate cells to be propagated between the clean bench 1 and the incubator 3, so that the apparatus disadvantageously becomes large.